Cell surface proteins constitute only a small fraction of the cellular proteins and these proteins are often not tumor-specific. Because of the inability to easily penetrate cells, marketed therapeutic monoclonal antibodies (mAbs) recognize these cell surface proteins, most of which are lineage or differentiation antigens (Milenic, E. D., Curr. Pharm. Des. 8:1794-1764, 2002; Grillo-Lopez, A. J., Expert Rev. Anticancer Ther. 2(3):323-329, 2002; Jones, K. L. & Buzdat, A. U., Lancet Oncol. 10(12):1179-1187, 2009). In contrast, mutated or oncogenic tumor-associated proteins are typically nuclear, cytoplasmic or secreted, which are currently best addressed either by small molecule drugs, or in the case of secreted proteins, hardly addressed as anti-cancer drug targets (Reddy et al., Expert Opin. Ther. Targets 3:313-324, 2012; Takeuchi, K. & Ito, F., Biol. Pharm. Bull. 34(12):1774-1780; Roychowdhury, S. & Talpaz, M., Blood Rev. 6:279-290, 2011). However, most intracellular proteins can be proteosomally degraded, processed and presented by MHC molecules on the cell surface as T cell peptide epitopes in the context of MHC molecules that are recognized by T cell receptors (TCRs) (Morris et al., Blood Rev. 20:61-69, 2006; Konnig, R., Curr. Opin. Immunol. 14(1):75-83, 2002). Therefore, generating therapeutic mAbs that recognize the secreted or intracellular tumor antigen-derived peptide/MHC complexes on the cell surface will take advantage of the enhanced specificity and therapeutic potency offered by mAbs. Recent advances in using phage display to generate mAbs have made it possible to select agents with exquisite specificity against defined epitopes from large antibody repertoires. A number of such mAbs specific for solid tumor antigens, in the context of HLA-A01 and HLA-A02, have been successfully selected from phage display libraries (Noy et al., Expert Rev. Anticancer Ther. 5(3):523-536, 2005; Chames et al., Proc. Natl. Acad. Sci. USA 97:7969-7974, 2000; Held et al., Eur. J. Immunol. 34:2919-2929, 2004; Lev et al., Cancer Res. 62:3184-3194, 2002; Klechevsky et al., Cancer Res. 68(15):6360-6367, 2008). More recently, a human mAb specific for human WT1/HLA-A02 complex, a well-described T cell epitope, has been shown to inhibit multiple cancer cell lines and primary cancer cells via Fc-mediated effector cell function (Dao et al., Sci. Transl. Med. 5:176ra33, 2013; Veomett et al., Clin. Cancer Res. doi: 10.1158/1078-0432, 2014) in cellular assays and in in vivo models.
Alpha-fetoprotein (AFP) is a 69 kD glycoprotein produced in the yolk sac and fetal liver and secreted into circulation. The synthesis of AFP decreases dramatically after birth and only trace amounts are present in the adult liver. In normal adult human blood serum, AFP concentration is usually as low as 5-7 ng/ml (Terentiev, A. A. & Moldogazieva, N. T., Tumour Biol. 34(4):2075-2091, 2013). However, expression of the AFP gene is reactivated in adults during liver regeneration, hepatocarcinogensis, germ cell tumor or in some cases of viral infection (HBV/HCV). The measurement of serum AFP, therefore, plays an important role in diagnosis and in monitoring responses to the treatment of AFP-positive cancers. Currently, AFP is considered a “gold standard” among tumor-specific molecular biomarkers. However, because AFP is not a cell-surface protein, targeting AFP has not been a very active area for anti-cancer antibody drug development.
Primary liver cancer is the fifth most common form of cancer worldwide and the second most common cause of cancer-related death. In 2012, there were about 782,000 new cancer cases globally, and about 746,000 liver cancer related deaths (http://globocan.iarc.fr/Pages/fact_sheets_cancer.aspx). 90-95% of liver cancers are hepatocellular carcinoma (HCC). Chronic liver damage, such as that caused by chronic hepatitis, liver cirrhosis and fatty liver disease, is closely associated with the occurrence of HCC. Hepatitis virus infection (e.g. HBV, HCV), aflatoxin B exposure, alcohol intake and other metabolic diseases (e.g. obesity and diabetes) are well-known risk factors for HCC. The incidence of HCC is high in East Asian and African countries due to the prevalence in HBV and HCV in these regions (Shibata, T. & Aburatani, H., Nat. Rev. Gastroenterol. Hepatol. 11(6):340-349, 2014). However, the number of patients infected with HCV has been rapidly increasing in Western countries, especially in the USA where viral hepatitis infection is partly mediated through drug abuse. Meanwhile, the incidences of cirrhosis owing to nonalcoholic steatohepatitis (NASH) and obesity also increased in Western countries. In the US, HCC is the 9th most common cancer (Vallanueva et al., Nat. Rev. Gastroenterol. Hepatol. 10(1):34-42).
Mean survival of patients with HCC is 3 months from diagnosis. However this is closely related to the stage of the tumor and the extent of underlying liver disease. As only a minority of HCC patients is considered suitable for resection and transplantation at the time of diagnosis, treatment for the majority of patients with HCC is mainly palliative. Non-surgical treatments, such as trans-arterial chemoembolization (TACE/TAE), radio-frequency ablation and systemic targeted agent like sorafenib, have been shown to reduce tumor burden and improve survival rate but these treatments do not eradicate cancer cells and the patients frequently relapse. Therefore, the development of more effective therapies remains a pressing field of research (Behboudi et al., Liver Int. 30(4):521-526, 2010).
AFP expression is reactivated in approximately 80% of HCC Immunotherapy studies aimed at generating AFP-specific cytotoxic CD8 T cell responses that recognize peptides presented on HCC cancer cell surface by MHC class I proteins have reported many human AFP peptides as T-cell epitopes (Butterfield et al., J. Immunol. 166:5300-5308, 2001; Pardee, A. D. & Butterfield, L. H., Oncolmmunol. 1:48-55, 2012; Butterfield et al., J. Trans. Med. 12:86, 2014; Liu et al., J. Immunol. 177 (1):712-721, 2006; Mizukoshi et al., Int. J. Cancer 118 (5):1194-1204, 2006). Among these peptides, FMNKFIYEI (AFP158) is an immunodominant T-cell epitope restricted by HLA-A*02:01. AFP/HLA-A*02:01 complex induced peptide-specific T cells in vitro from normal HLA-A*02:01 donors. These AFP158 specific T cells recognized HLA-A*02:01 positive and AFP positive tumor cells in both cytotoxicity assays and IFNγ ELISPOT assays. AFP158 was identified by mass spectrometric analysis of surface peptides from an HLA-A*02:01 positive HCC cell line, HepG2, but not from a HLA-A*02:01 negative cancer cell line, Hep3B (Butterfield et al., J. Immunol. 166:5300-5308, 2001). These data support that AFP158 is indeed processed and presented by HLA-A*02:01 molecules in AFP-positive cancer cells. Therefore, AFP158/HLA-A*02:01 is a good target candidate for mAb cancer drug development.
Traditional approaches to using AFP as a therapeutic target for treating cancers that overexpress AFP have relied on using antibodies that target the AFP protein or vaccination with various AFP peptides. Antibodies directed against the AFP protein have little therapeutic efficacy since AFP is not a cell-surface protein, and may be present at high circulating levels, thus reducing any specific targeting of the antibody to the cells expressing AFP. While vaccination with various MHC-restricted AFP peptides and variants thereof has been observed to lead to activation of AFP-specific T cells and increased cytotoxicity of AFP-presenting cells in vitro, a clinically significant therapeutic benefit has yet to be observed.
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